Wnt signaling plays an important role in animal development, and misregulation of the pathway contributes to various human diseases, including a variety of cancers. The proposed experiments are designed to identify and characterize regulators of the homologous Wingless (Wg) signaling pathway in Drosophila. Though many of the core components of the pathway have been identified, factors involved in the cell-specific effects of Wg signaling on gene expression patterns are still poorly understood. The experiments described in this proposal focus on elucidating how Wg signaling alters gene expression in various tissues through the action of the transcriptional coactivator, Armadillo (Arm). To identify regulators of Arm transcriptional activity, we performed a small-scale genetic screen and we identified 15 potential regulators of Arm activity. One gene identified in the screen was mastermind (Mam), a previously characterized coactivator in the Notch signaling pathway. The first two aims of this proposal are focused on testing the hypothesis that Mam-RC, a naturally occurring short isoform of Mam, is a tissue-specific regulator of Arm activity. Experiments outlined in Specific Aim 1 are designed to determine the molecular mechanism by which Mam-RC regulates Arm transcriptional activity. We propose cell culture and biochemical experiments that will test for both physical and functional interactions of Mam-RC with known components of the Arm transcription complex. Specific Aim 2 outlines experiments designed to test the hypothesis that Mam-RC is a tissue-specific regulator of Wg signaling in vivo, by manipulating the levels of mam-RC expression in various tissues in the developing fly. In addition to Mam-RC, we identified other possible regulators of Arm; Specific Aim 3 outlines experiments designed to characterize the role of these other genes in regulating Arm both in vivo and in cell culture. Given the evolutionary conservation of the pathway components in all animals, findings from our studies of these novel regulators of Arm-dependent transcription may provide greater insight into both human development and disease.